TOP10 chemically competent cells

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(Preparing competent cells)
Line 40: Line 40:
* 10 mM MgCl<sub>2</sub>.6H<sub>2</sub>O (2.0 g/l)
* 10 mM MgCl<sub>2</sub>.6H<sub>2</sub>O (2.0 g/l)
* 10% glycerol (100 ml/l)
* 10% glycerol (100 ml/l)
-
* adjust pH to 6.4 with 0.1N HCl
+
* adjust pH DOWN to 6.4 with 0.1N HCl if necessary
** adjusting pH up will precipitate manganese dioxide from Mn containing solutions.
** adjusting pH up will precipitate manganese dioxide from Mn containing solutions.
* sterile filter and store at 4C.
* sterile filter and store at 4C.
Line 49: Line 49:
** This is at 10 pg/&mu;l or 10<sup>-5</sup> &mu;g
** This is at 10 pg/&mu;l or 10<sup>-5</sup> &mu;g
* Hold on ice 0.5 hours
* Hold on ice 0.5 hours
-
* Heat shock 60 sec
+
* Heat shock 60 sec at 42C
* Add 250 &mu;l SOC
* Add 250 &mu;l SOC
* Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated
* Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated
 +
** using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.
* Plate 20 &mu;l on AMP plates using 3.5 mm glass beads
* Plate 20 &mu;l on AMP plates using 3.5 mm glass beads
* Transformation efficiency is 15 x colony count x 10<sup>5</sup>
* Transformation efficiency is 15 x colony count x 10<sup>5</sup>

Revision as of 09:28, 27 July 2006

This protocol is a variant of the Hanahan protocol [1] using CCMB80 buffer for DH10B and TOP10 strains. It builds on Example 2 of the Bloom05 patent as well.

Contents

Preparing glassware and media

Detergent is a major inhibitor of competent cell growth and transformation. Glass and plastic must be detergent free for these protocols. The easiest way to do this is to avoid washing glassware, and simply rinse it out. Autoclaving glassware filled 3/4 with DI water is an effective way to remove most detergent residue. Media and buffers should be prepared in detergent free glassware and cultures grown up in detergent free glassware.

Preparing seed stocks

  • streak TOP10 cells on an SOB plate and grow for single colonies at 23 C
    • room temperature works well
  • Pick single colonies into 2 ml of SOB medium and shake overnight at 23 C
    • room temperature works well
  • Add glycerol to 15%
  • Aliquot 1 ml samples to Nunc cryotubes
  • Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes
    • This step may not be necessary
  • Place in -80 freezer indefinitely.

Preparing competent cells

  • Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20 C to an OD of 0.5
    • This takes approximately 16 hours. Controlling the temperature makes this a more reproducible process, but is not essential. Room temperature will work. You can adjust this temperature somewhat to fit your schedule
  • Centrifuge at 3000g / 4C for 10 minutes in a flat bottom centrifuge bottle.
    • Flat bottom centrifuge tubes make the fragile cells much easier to resuspend
  • Gently resuspend in 80 ml of ice cold CCMB80 buffer
  • Incubate on ice 20 minutes
  • Centrifuge again at 4C and resuspend in 20 ml of ice cold CCMB80 buffer.
  • Incubate on ice for 20 minutes
  • Aliquot to chilled screw top 2 ml vials or 50 μl into chilled microtiter plates
  • Store at -80C indefinitely.
    • Flash freezing does not appear to be necessary


CCMB80 buffer:

  • 10 mM KOAc pH 7.0 (10 ml of a 1M stock/l)
  • 80 mM CaCl2.2H2O (11.8 g/l)
  • 20 mM MnCl2.4H2O (4.0 g/l)
  • 10 mM MgCl2.6H2O (2.0 g/l)
  • 10% glycerol (100 ml/l)
  • adjust pH DOWN to 6.4 with 0.1N HCl if necessary
    • adjusting pH up will precipitate manganese dioxide from Mn containing solutions.
  • sterile filter and store at 4C.


Measurement of competence:

  • Transform 50 μl of cells with 1 μl of standard pUC19 plasmid (Invitrogen)
    • This is at 10 pg/μl or 10-5 μg
  • Hold on ice 0.5 hours
  • Heat shock 60 sec at 42C
  • Add 250 μl SOC
  • Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated
    • using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.
  • Plate 20 μl on AMP plates using 3.5 mm glass beads
  • Transformation efficiency is 15 x colony count x 105

References

  1. Hanahan D, Jessee J, and Bloom FR. . pmid:1943786. PubMed HubMed [Hanahan91]
  2. US Patent 6,855,494 Media:pat6855494.pdf [Bloom05]


6/7/06 colony counts:

  • Invitrogen control: 281
  • M : 54
  • S1: 125
  • 15/1: 74
  • S2: 166
  • 15/2: 177
  • S3: 243
  • 15/3: 60

15/ cells are grown in 15/10 medium. Slow growth, eliminate this idea. Dispose of M, 15/1, 15/2, 15/3 cells. Redoing competent cells today. m/s1/s2/s3 cells are successive versions of competent cells grown in SOB medium.

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